The present invention relates to the construction of a specific type of probe to be used in the detection of cryptic viral infections of plants at an early stage before the infections have extensively spread through agricultural fields. If such infections are detected early, the spread of such viral infections can be slowed or even stopped by known methods which inclue spraying insecticides.
Alfalfa mosaic virus (AMV) is one of a family of plant viruses (the Tricornaviridae) with a single stranded plus type RNA genome. The genome (excluding the subgenomic RNA molecules) is segmented into three RNA molecules. This family is defined to include: the alfalfa mosaic virus (AMV) group (of which AMV is presently the only member), the ilarviruses, the bromoviruses and the cucumoviruses (van Vloten-Doting, L., R. I. B. Francki, R. W. Fulton, J. M. Kaper and L. C. Lane (1981) Intervirology 15: 198-203). The genome fragments (bottom (B)-, middle (M)-, and top component b(Tb)-RNA) are separately encapsidated in bacilliform particles of different lengths. Besides these three components, a fourth particle (top component a (Ta)) containing two identical sized RNA molecules is found in virus preparations. A mixture of the three genome fragments ((B))-, (M)- and (Tb)-RNA) together with a small amount of coat protein or its messenger, Ta-RNA (Bol, J. F., van Vloten-Doting, L. and Jaspars, E. M. J. (1971) Virology 46: 73-85) is required to initiate infection. Coat protein occurs in polyribosomes extracted from infected leaves and in preparations of the soluble virus replicase, thus indicating that the coat protein has a regulatory function in the translation and/or replication of virus RNA. There is a high degree of homology in the 145 base pairs at the 3'-termini of all four RNAs. (C. J. Houwing and E. M. J. Jaspars (1978) Biochemistry 17: 2927-2933).
The B-, M-, Tb- and Ta-RNA genome fragments are also referred to as RNA 1, RNA 2, RNA 3 and RNA 4, respectively. The complete sequence of AMV RNA 4 has been described (F. T. Brederode, E. C. Koper-Zwarthoff and J. F. Bol (1980) Nucleic Acids Res. 8: 2213-2223). RNA 4 is 881 nucleotides in length. The coding region is 660 nucleotides (not including the initiation and termination codon) flanked by a 5'-noncoding region of 39 nucleotides and a 3'-noncoding region of 182 nucleotides. The sequence of RNA 4 is present in RNA-3 and located at the 3'-end of this RNA species (Gould, A. R. and Symons, R. H. (1978) Eur. J. Biochem. 91: 269-278).
In addition, the complete nucleotide sequence of alfalfa mosaic virus RNA 1 has been published (B. J. C. Cornelissen, F. T. Brederode, R. J. M. Moormann and J. F. Bol (1983) Nucleic Acids Res. 11: 1253-1265). Double stranded cDNA was cloned and the sequence data were obtained from clones with overlapping inserts. The complete sequence is 3645 nucleotides in length and it contains a long open reading frame for a protein of Mw 125,685 flanked by a 5'-terminal sequence of 99 nucleotides and a 3'-noncoding region of 163 nucleotides, including the sequence of 145 nucleotides which the three genomic RNAs of AMV have in common.
A limited amount of information is available on the 5'-terminal and 3'-terminal sequences of AMV RNA 2 and 3. These sequences were obtained by sequencing RNA strands. At the 5'-termini, the sequence of 13 nucleotides of AMV RNA 2 and 101 nucleotides of AMV RNA 3 have been published (E. C. Koper-Zwarthoff, F. T. Brederode, G. Veeneman, J. H. van Boom and J. F. Bol (1980) Nucleic Acids Res. 8: 5635-5647). Extensive homology occurs between the first 11 nucleotides of all four AMV RNAs. AMV RNA 3 is dicistronic and the sequence of 122 nucleotides at the intercistronic junction is known (E. C. Koper-Zwarthoff et al. (1980) Nucleic Acids Res. 8: 5635-5647). Finally the sequences of 227 nucleotides at the 3'-terminus of RNA 3 and of 169 nucleotides at the 3'-terminus of RNA 2 are known (E. C. Zwarthoff, F. T. Brederode, P. Walstra and J. F. Bol (1979) Nucleic Acids Res. 7: 1887-1900). A. R. Gould and R. H. Symons ((1978) Eur. J. Biochem. 91: 269-278) presented evidence that the sequence of AMV RNA 4 is located at the 3'-end of RNA 3.
A comparison of the 3'-terminal sequences of the genomic AMV RNAs (RNA 1, RNA 2 and RNA 3) and of the sub-genomic RNA (RNA 4) has revealed extensive homology between the 3'-terminal 140 to 150 nucleotides of all four RNAs. There are about 20 base substitutions in the 3'-terminal 145 nucleotides of the AMR RNAs but these are either located in the loops of base paired structures or convert A-U base pairs to G-C base pairs in the stems of the secondary structure hairpins (E. C. Koper-Zwarthoff, F. T. Brederode, P. Walstra and J. F. Bol (1979) Nucleic Acids Res. 7: 1887-1900).
RNA sequencing methods are inferior to DNA sequencing methods and so cDNA cloning is a critical tool in molecular biological research. Considerable effort has therefore been made towards optimizing conditions for the preparation of full length cDNA clones. The original approaches to cDNA cloning (Efstradiadis, A., Kafatos, F. C., Maxam, A. M. and Maniatis, T. (1976) Cell 7: 279-288; Rougeon, F. and Mach, B. (1976) Proc. Nat'l. Acad. Sci. U.S.A. 73: 3418-3422) are still in widespread use. By these methods the synthesis of the anticomplementary strand is assumed to be dependent on the formation of a hairpin at the 3'-end of the complementary strand. This hairpin must then be removed by S1 nuclease thereby giving an incomplete cDNA. This is particularly important since the treatment degrades the cDNA corresponding to the 5'-end of the mRNA. Consequently, aspects of an alternate approach originally developed by Rougeon and Mach, supra, which avoids the use of S1 nuclease have been used recently to obtain improved yields of full length clones with intact 5'-ends (Land, H., Grez, M., Hauser, H., Lindemaier, W. and Schutz, G. (1981) Nucleic Acids Research 9: 2251-2266 and Okayama, H. and Berg, P. (1982) Molec. and Cell Biology 2: 161-170). This is achieved by adding a homopolymer tail to the 3'-end of the complementary strand and priming the second strand with the complementary oligonucleotide.
The full implications of the fact that reverse transcriptase can, during the synthesis of the complementary DNA strand, also synthesize the anticomplementary strand (D. L. Kacian and J. C. Meyers (1976) Proc. Nat'l Acad. Sci. U.S.A. 73: 3408-3412) are not generally considered in most cloning schemes. Anticomplementary synthesis has been shown to be largely attributable to an RNAse H activity which resides on the same enzymatic subunit as does the RNA-dependent DNA polymerizing activity (D. P. Grandgennet, G. F. Gerrard and M. G. Green (1973) Proc. Nat'l Acad. Sci. U.S.A. 70: 230-234 and J. C. Meyers, C. Dobkin and S. Spiegelman (1980) Proc. Nat'l Acad. Sci. U.S.A. 77: 1316-1320). After a cDNA-RNA duplex has been formed by the DNA polymerizing activity, the RNA is nicked and degraded into oligoribonucleotides by the RNAs H activity. These oligoribonucleotides are thought to prime the DNA-dependent synthesis of the anticomplementary strand using the complementary strand as a template. Because such priming events occur randomly, the anticomplementary strand consists of a discontinuous series of segments. The inclusion of 4 mM sodium pyrophosphate (NaPPi) in reverse transcriptase reactions reportedly yields longer DNAs. NaPPi has been reported to inhibit RNAse H activity to some extent but does not appear to affect the length of the complementary strand (D. L. Kacian and J. C. Meyers (1976), supra, and J. C. Meyers and S. Spiegelman ( 1978) Proc. Nat'l Acad. Sci. U.S.A. 75: 5329-5333). Actinomycin D has also been used to inhibit anticomplementary DNA synthesis; however, it has been reported to be less effective than NaPPi as an inhibitor of RNAase H activity. (D. L. Kacian and J. C. Meyers (1976), supra, and G. De Martynoff, E. Pays and G. Vassart (1980) Biochem. Biophys. Res. Comms. 93: 645-653).
An important consequence of unanticipated anticomplementary DNA synthesis is that it can seriously reduce the probability of obtaining full length cDNA clones if the products of the presumptive first strand reaction (i.e., complementary and anticomplementary DNA) are denatured and submitted to a second synthesis reaction. In such cases, short double stranded DNAs resulting when the anticomplementary strands are rendered double stranded will inevitably outnumber any full anticomplementary strand synthesis. The present invention describes methods whereby these prior art difficulties in obtaining full length cDNA clones are largely overcome.
DNA sequences have been used extensively to probe for specific RNA sequences. For example, the insulin gene has been cloned in a recombinant plasmid and used to detect the presence of insulin mRNA in pancreatic tissue. In another instance cloned early histone genes of sea urchins have been used to detect the presence of specific histone mRNA molecules in various stages of sea urchin embryogenesis. Thus the availability of a specific deoxynucleotide sequence can aid in the detection of a specific RNA either in different organs or at different stages of the life cycle. The specific DNA sequence can be easily maintained and propagated as part of a recombinant plasmid in a bacterial strain.
More specifically, cloned DNA sequences have been used to detect the presence of RNA molecules in plant tissue. For example, cDNA clones have been constructed using mRNA purified from developing endosperms of maize. These cDNA clones have been used to determine relative levels of specific maize zein mRNAs transcribed during maize endosperm development (M. D. Marks and B. A. Larkins (1983) J. Cell. Biochem. Suppl. 7B: 278 12th Annual UCLA Symposia). In a different example, using poly(A) RNA from French bean (Phaseolus vulgaris), a cDNA library was constructed and screened with pea lectin cDNA to yield a clone coding for an entire mature lectin peptide. By use of this cloned cDNA, the ontogeny of lectin gene expression in several plant tissues has been studied (L. M. Hoffman (1983) J. Cell Biochem. Suppl. 7B: 279 12th Annual UCLA Symposia).
A sensitive and reliable new method based on hybridization of highly radioactive cDNA to potato spindle tuber viroids (PSTV) has been described (R. A. Owens and T. O. Diener (1981) Science 213: 670-672). The PSTV can be bound to a solid support (e.g., nitrocellulose membrane) and its presence can be detected by autoradiography with the labelled cDNA. Comparison of relative autoradiographic intensities showed that the presence of sap from uninfected tuber sprouts reduced the binding approximately tenfold, but 83-250 .mu.g of PSTV were still easily detectable after hybridization with radioactive cDNA. This amount is equivalent to a concentration of 0.04-0.125 .mu.g PSTV per gram of tuber sprouts. Actively growing potato tissue contains 0.5 .mu.g or more PSTV per gram of tissue (M. A. Pfannenstiel, S. A. Slack and L. C. Lane (1980) Phytopathology 70: 1015). The hybridization method described was therefore adequate to detect PSTV in potato tissue.
A recombinant DNA cloning experiment involves a number of essential steps. Firstly, there must be a method for generating DNA fragments whose ends can be ligated into vector DNA molecules. Secondly, this composite, recombined DNA molecule must be introduced into a host cell in which it can be replicated, i.e., cloned. Thirdly, since a very large number of genes exist in an organism, a method must be found for detecting a specific nucleotide sequence that has been inserted into a vector DNA of a specific recombinant DNA clone. One method has been to isolate mRNA molecules and from these to generate double-stranded complementary DNA (cDNA). In many cases the isolation of a cDNA has been facilitated by using tissues in which specific genes are functional. Thus a large proportion of mRNAs in reticulocyte cells are globin mRNAs and a large proportion of mRNAs from pancreatic islet cells are insulin mRNAs. The majority of mRNAs are polyadenylated at the 3'-ends subsequent to transcription, and this feature can be conveniently used to generate cDNA from mRNA. Oligo(dT), used as a primer, will anneal to the poly(A) tail and reverse transcriptase in the presence of all four deoxynucleoside triphosphates can be used to replicate a complementary single-stranded DNA on the mRNA.
The first product is thus an RNA-DNA hybrid. The RNA strand can then be destroyed by alkaline hydrolysis, to which DNA is resistant, leaving a single-stranded cDNA which can be converted into the double-stranded form in a second DNA polymerase reaction. This reaction depends upon the observation that single-stranded cDNA's can form a transient self-priming structure in which a hairpin loop at the 3' terminus is stabilized by enough base pairing to allow initiation of second strand synthesis. Once initiated, subsequent synthesis of the second strand stabilizes the hairpin. The hairpin is then trimmed away by treatment with the single strand specific nuclease S1, giving rise to a fully duplex molecule which has lost several nucleotide residues corresponding to the 5'-terminus of the RNA strand. This fact serves to emphasize that most cDNAs derived from mRNAs lack portions of the 5'-untranslated termini and that the term "full length" has frequently been used in the prior art to refer to the complete coding region, rather than the complete mRNA nucleotide sequence.
This cDNA molecule can then be tailed with oligo(dC) and annealed with a vector (e.g., pBR322) which has been cut open with a restriction endonuclease (e.g., Pst I) and tailed with oligo(dG). (For further general discussion see Old, R. W. and Primrose, S. B. Principles of gene manipulation--an introduction to genetic engineering (1980) University of California Press). A wide variety of DNA vectors known in the art may be employed as cloning vehicles. The choice of DNA vector to be employed will depend upon considerations known to those of ordinary skill in the art, such as the desired insertion site, selection means, stability and the like. A DNA vector into which cDNA has been inserted is termed herein a recombinant DNA plasmid. The recombinant cDNA plasmid is then transformed into a suitable bacterial host strain where it can be propagated.
The majority of mRNAs in eukaryotes have polyadenylated 3'-tails. In contrast, RNAs of many plant viruses do not have poly-A tails. Thus, in order to construct cDNA's of the plant virus RNA's, it is first necessary to add a poly-A tail. (Sippel, A. E. (1973) Eur. J. Biochem. 37, 31-40).
The cloned cDNA is then multiplied in the bacterial strain. After purification, the cDNA probe is labelled with .sup.32 P to high specific activity (about 10.sup.8 cpm/.mu.g) by method known in the art, for example, nick translation (P. W. Rigby, M. Dieckmann, C. Rhodes and P. Berg (1977) J. Mol. Biol. 113: 237). This highly labelled cDNA probe enables detection of restriction fragments of DNA derived from single copy genes or from an infection level of one virus particle per cell. In order to detect specific DNA fragments or RNA molecules, the DNA fragments or RNA molecules are first bound by known methods in a denaturing solution to a solid phase material such as nitrocellulose membranes (E. M. Southern (1975) J. Mol. Biol. 98: 503-517 and P. S. Thomas (1980) Proc. Nat'l Acad. Sci. U.S.A. 77: 5201). The labelled cDNA probe can then also be denatured and added in a re-annealing solution to the surface of the nitrocellulose membrane under conditions where cDNA does not bind to the membrane itself. If the cDNA is complementary to the genomic DNA and to the RNA molecules, it will reanneal to the relevant sequences. The unbound labelled cDNA can then be washed away and the labelled fragments described by radioautography. By this method, fragments of genomic DNA containing specific gene sequences (e.g., globin genes) or specific RNAs (e.g., sea urchin histone mRNAs in embryogenesis) can be identified.